Numerous neuronal ultrastructural and biochemical abnormalities develop during brain ischemia and reperfusion; however, the causal interrelationships of these phenomena are not well understood. Dr.Neumar has recently found that (1) brain eIF-4E levels decline sharply during ischemia and (2) eIF-4E is rapidly degraded in vitro by Ca2+-activated mu- calpain. Proteolysis of eIF-4E during ischemia could be due to calcium- induced mu-calpain activation. mRNA binding by eIF-4E is a rate limiting step in translation initiation, and degradation of eIF-4E by mu-calpain could connect the recognized phenomena of calcium influx during ischemia and subsequent depressed protein synthesis. The hypotheses proposed are that (1) ischemia in vivo and Ca2+ overloading in vitro cause mu-calpain activation and eIF-4E degradation in neurons, (2) ischemia-induced activation of mu-calpain and loss of eIF-4E are most prominent in selectively vulnerable neurons, and (3) pretreatment with a calpain inhibitor reduces both mu-calpain autoproteolytic activation and degradation of eIF-4E. Dr. Neumar will study these hypotheses utilizing ((i) an in vivo model of complete brain ischemia and reperfusion by cardiac arrest and resuscitation in rats and (ii) inophore-induced Ca2+ overload in neuronally-differentiated NB-104 cells. Antibodies specific to eIF-4E and activated mu-calpain will be used in Western blots to characterize (i) in cultured neurons Ca2+-induced and (ii) in rat brains ischemia and reperfusion-induced mu-calpain activation and eIF-4E degradation. Light and electron microscopy with immunohistochemistry will provide regional and ultrastructural localization of mu-calpain activation and eIF-4E degradation with simultaneous ultrastructural evaluation for neuronal injury. The effect of a cell-permeant mu-calpain inhibitor will be evaluated in all of the above experiments.